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J Biol Chem, Vol. 273, Issue 15, 8553-8555, April 10, 1998

COMMUNICATION
Cryo-crystallography of a True Substrate, Indole-3-glycerol Phosphate, Bound to a Mutant (D60N) Tryptophan Synthase ₂₂ Complex Reveals the Correct Orientation of Active Site Glu49

Sangkee Rhee, Edith Wilson Miles^§, and David R. Davies

From the  Laboratory of Molecular Biology and the ^§ Laboratory of Biochemistry and Genetics, NIDDK, National Institutes of Health, Bethesda, Maryland 20892

The reversible cleavage of indole-3-glycerol by the -subunit of tryptophan synthase has been proposed to be catalyzed by Glu49 and Asp60. Although previous x-ray crystallographic structures of the tryptophan synthase ₂₂ complex showed an interaction between the carboxylate of Asp60 and the bound inhibitor indole-3-propanol phosphate, the carboxylate of Glu49 was too distant to play its proposed role. To clarify the structural and functional roles of Glu49, we have determined crystal structures of a mutant (D60N) ₂₂ complex in the presence and absence of the true substrate, indole-3-glycerol phosphate. The enzyme in the crystal cleaves indole-3-glycerol phosphate very slowly at room temperature but not under cryo-conditions of 95 K. The structure of the complex with the true substrate obtained by cryo-crystallography reveals that indole-3-glycerol phosphate and indole-3-propanol phosphate have similar binding modes but different torsion angles. Most importantly, the side chain of Glu49 interacts with 3-hydroxyl group of indole-3-glycerol phosphate as proposed. The movement of the side chain of Glu49 into an extended conformation upon binding the true substrate provides evidence for an induced fit mechanism. Our results demonstrate how cryo-crystallography and mutagenesis can provide insight into enzyme mechanism.

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Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.

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